Molecular bases of daptomycin-resistance-mediated sensitization to ? lactams (see-saw effect) in clinical MRSA strains Methicillin-resistant Staphylococcus aureus (MRSA) is an important infectious human pathogen for which treatment options are very limited. MRSA strains have acquired and developed many antibiotic resistance mechanisms. Daptomycin (DAP) is a cyclic anionic lipopeptide antibiotic produced by Streptomyces roseosporus. It was approved by the FDA in 2003 for use in a wide variety of S. aureus infections. The mechanism of action of DAP involves the disruption of cytoplasmic membrane function leading to its depolarization and causing cell death. Due to its potent staphylocidal activity, DAP has become the clinical mainstay of anti-MRSA therapy, especially in patients with persistent bacteremia. However, there have been a number of reports in which initially DAP-susceptible (DAPS) MRSA strains developed DAP resistant (DAPR) phenotypes and resulted in clinical treatment failures. DAP resistance mechanisms have been associated mostly with alterations in cell membrane. Our data suggest that both cell membrane and cell wall factors are implicated in DAP resistance acquisition. We have demonstrated that in addition to mprF (membrane gene), DAP resistance involved upregulation of genes involved in cell wall synthesis and turnover, including the two-component regulator and cell wall stress stimulon vraSR. Interestingly, we and others have observed that DAP resistance sensitizes MRSA to oxacillin, a process known as a see-saw effect; however, the mechanisms remain speculative and have not yet been elucidated. Altogether, these observations led to our main hypothesis, i.e. that DAP-mediated changes in the cell membrane affect cell wall formation and proteins directly involved in its synthesis and turnover, re-sensitizing these strains to cell wall-specific beta lactams. This proposal intends to elucidate the molecular bases of DAP resistance-mediated sensitization to beta-lactams. The Specific Aims of this revised version are: Specific Aim #1: To investigate cell membrane/cell wall factors involved in DAP-resistance-mediated sensitization to beta-lactams (see-saw effect). Specific Aim #2 To establish the role of penicillin binding proteins (PBP)-1, -2 and PBP2a during the DAP-mediated see-saw effect. Specific Aim #3: To determine the mutual regulatory interactions between cell membrane-associated mprF and cell wall-related two-component regulator vraSR. Specific Aim #4 To analyze the efficacy of DAP/beta-lactam combination for treatment of clinically relevant DAP- susceptible/-resistant MRSA strains. We believe the proposed studies can shed light on the mechanistic bases that will lead to improved treatments against one of the most important health issues in infectious disease control.